Differentiation by molecular typing of Mycobacterium bovis strains causing tuberculosis in cattle and goats.

Forty Mycobacterium bovis isolates from cattle and goats were analyzed by using different repetitive genetic markers. The 23 M. bovis strains from goats were found to carry six to eight copies of the insertion sequence IS6110. In contrast, most of the bovine isolates contained only a single copy of this element. The standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP), described for Mycobacterium tuberculosis strains, allowed the differentiation of caprine strains. Although this method was not useful for typing bovine isolates, the repetitive elements pTBN12 and DR proved to be suitable for this purpose. A procedure using PCR which amplifies IS6110 in the outward direction was found to be as sensitive as RFLP for typing M. bovis strains from goats. The use of PCR and RFLP methods based on the IS6110 polymorphism would be useful for epidemiological studies of caprine tuberculosis. The results are consistent with different strains of M. bovis being implicated in bovine and caprine tuberculosis.     

Evaluation of method for secondary DNA typing of Mycobacterium tuberculosis with pTBN12 in epidemiologic study of tuberculosis.

Secondary fingerprinting of Mycobacterium tuberculosis DNA with a probe containing the polymorphic GC-rich repetitive sequence present in pTBN12 has been found to have greater discriminating power than does fingerprinting with the insertion sequence IS6110 for strains carrying few copies of IS6110. To validate the use of pTBN12 fingerprinting in the molecular epidemiology of tuberculosis, M. tuberculosis isolates from 67 patients in five states in the United States and in Spain were fingerprinted with both IS6110 and pTBN12. Epidemiologic links among the 67 patients were evaluated by patient interview and/or review of medical records. The 67 isolates had 5 IS6110 fingerprint patterns with two to five copies of IS6110 and 18 pTBN12 patterns, of which 10 were shared by more than 1 isolate. Epidemiologic links are consistently found among patients whose isolates had identical pTBN12 patterns, whereas no links were found among patients whose isolates had unique pTBN12 patterns. This suggests that pTBN12 fingerprinting is a useful tool to identify epidemiologically linked tuberculosis patients whose isolates have identical IS6110 fingerprints containing fewer than six fragments.     

Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis.

A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more copies of IS6110. Analysis of isolates with unique IS6110 fingerprints demonstrated that they were unique with pTBN12. The pTBN12 probe had greater discriminating power in isolates having five or fewer copies of IS6110. Forty-seven isolates from Denver having fewer than five copies of IS6110 which were grouped in 11 clusters with identical fingerprint patterns were subdivided into 35 different patterns by pTBN12. Isolates with IS6110 fingerprints with more than six copies of IS6110 that differed from one another by only one or two hybridizing bands were analyzed with pTBN12. Most of these sets of isolates demonstrated identical patterns with pTBN12. However, some exceptions were observed, suggesting that those having nearly identical IS6110 patterns should not necessarily be included in the same cluster. Since IS6110 provides more polymorphism in the fingerprint, it is most useful in identifying isolates with unique fingerprint patterns and those in clusters in which the isolates contain six or more copies of the insertion. However, it is necessary to employ a secondary probe, such as pTBN12, to discriminate isolates with five or fewer copies of IS6110 and those with similar but not identical IS6110 patterns.     

Differentiation of Mycobacterium bovis isolates from animals by DNA typing.

The insertion sequence IS6110 and the direct repeat (DR) specific to tuberculosis complex mycobacteria and the highly repeated DNA sequence, the polymorphic GC-rich repeat sequence (PGRS), were systematically used to identify restriction fragment length polymorphisms (RFLPs) within 210 isolates of Mycobacterium bovis. The isolates were primarily of bovine origin, but isolates from badgers, feral deer, sheep, humans, and a pig were included. The RFLP probes IS6110, DR, and PGRS individually identified 17, 18, and 18 different RFLP types, respectively, but in combination these probes identified a total of 39 different M. bovis RFLP types. The recommendations (J. D. A. van Embden, M. D. Cave, J. T. Crawford, J. W. Dale, K. D. Eisenach, B. Gicquel, P. W. M. Hermans, C. Martin, R. McAdam, T. M. Shinnick, and P. M. Small, J. Clin. Microbiol. 31:406-409, 1993) for a standardized RFLP analysis for M. tuberculosis were adapted to facilitate gel documentation, image analysis, and construction of a database of RFLP types. In the present study the same M. bovis RFLP types were evident in the various animal species included, indicating that the strains were not host restricted. Application of these techniques to defined field studies should help elucidate more accurately aspects of the epidemiology of bovine tuberculosis in different countries.     

Spoligotyping and polymorphic GC-rich repetitive sequence fingerprinting of mycobacterium tuberculosis strains having few copies of IS6110

Several genetic loci have been utilized to genotype isolates of Mycobacterium tuberculosis. A shortcoming of the most commonly used method, IS6110 fingerprinting, is that it does not adequately discriminate between isolates having few copies of IS6110. This study was undertaken to compare pTBN12 fingerprinting of polymorphic GC-rich repetitive sequence genes and spoligotyping of the direct repeat locus as secondary typing procedures for M. tuberculosis isolates having fewer than six copies of IS6110. A total of 88 isolates (100% of the isolates with fewer than six copies of IS6110 isolated in Arkansas during 1996 and 1997) were included in this study. Among the 88 isolates, 34 different IS6110 patterns were observed, 10 of which were shared by more than 1 isolate, involving a total of 64 isolates. The 64 isolates were subdivided into 13 clusters (containing 37 isolates) and 27 unique isolates based on a combination of IS6110 and pTBN12 fingerprinting and into 11 clusters (containing 51 isolates) and 13 unique isolates based on a combination of IS6110 fingerprinting and spoligotyping. Identical spoligotypes were found among isolates having different IS6110 patterns, as well as among isolates showing different pTBN12 patterns. In contrast, all isolates that had different IS6110 patterns were found to be unique by pTBN12 typing. The clustering rate was 73, 58, and 42%, respectively, for IS6110 fingerprinting alone, IS6110 fingerprinting and spoligotyping combined, and IS6110 and pTBN12 combined fingerprinting. The data indicate that the pTBN12 method has greater discriminating power among low-copy-number isolates than does spoligotyping.     

DNA fingerprinting of Mycobacterium bovis strains by restriction fragment analysis and hybridization with insertion elements IS1081 and IS6110.

Strains of Mycobacterium bovis, the causative organism of bovine tuberculosis, can be clearly distinguished from each other by restriction fragment analysis. This method of DNA fingerprinting has been used for many epidemiological studies in New Zealand, but the technique presents practical difficulties that hinder its widespread use. The insertion element IS6110 is being widely used as a DNA probe for distinguishing restriction fragment polymorphisms among strains of Mycobacterium tuberculosis. Both this element and another recently sequenced element, IS1081, are also present in M. bovis. We assessed the usefulness of these two elements for distinguishing between 160 strains of M. bovis. These strains, most of which were isolated in New Zealand, were selected to be representative of the 95 different types that were identified among 530 strains that were previously typed by restriction fragment analysis. Fifteen IS6110 types were identified, but more than half of the strains representing 46 restriction types had the same IS6110 type. Virtually all M. bovis strains as well as strains of M. tuberculosis and Mycobacterium africanum had the same IS1081 type. The results indicate that for M. bovis, IS1081 cannot be used to type strains, IS6110 can be used to distinguish strains into broad groups, but only restriction fragment analysis is sufficiently sensitive for detailed epidemiological studies. An investigation of the host range of IS1081 revealed that, apart from its presence in species of the tuberculosis complex, it is also present in a strain of Mycobacterium xenopi.     

Identification of a novel DNA probe for strain typing Mycobacterium bovis by restriction fragment length polymorphism analysis

Bovine tuberculosis caused by Mycobacterium bovis remains a significant disease of farmed cattle in many countries despite ongoing tuberculosis eradication programs. Molecular typing methods such as restriction fragment length polymorphism (RFLP) analysis and spoligotyping have been used to identify related herd breakdowns in an attempt to identify more precisely the route of infection into cattle herds and to trace the transmission of bovine tuberculosis. A recent geographical survey of Irish M. bovis isolates demonstrated that a significant proportion of isolates ( approximately 20%) exhibit a common strain type, limiting the value of current strain typing methods as an epidemiological tool. We have identified and cloned a region of the M. bovis genome, pUCD, which generates a clear, highly polymorphic banding pattern when used as an RFLP probe on AluI restriction-digested M. bovis genomic DNA and which effectively subdivides this common strain type. When used to type 60 Irish M. bovis isolates, pUCD exhibited greater discriminatory power than the commonly used mycobacterial RFLP probes IS6110, PGRS, and DR and detected an equivalent number of strain types to a combination of these three probes. pUCD also detected significantly more strain types than the spoligotyping technique, while maintaining a high level of concordance between epidemiologically related and unrelated herd breakdowns. The polymorphic element within pUCD remains to be fully characterized, however the potential for this probe to greatly decrease the workload necessary to genotype M. bovis by RFLP analysis is compelling.     

Monitoring of Transmission of Tuberculosis between Wild Boars and Cattle: Genotypical Analysis of Strains by Molecular Epidemiology Techniques

An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen Mycobacterium bovis strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15 Mycobacterium bovis strains were subsequently cultured. All M. bovis strains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS6110 and IS1081 probes. All M. bovis strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in M. tuberculosis. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS6110 probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS1081 as a second probe was useful for differentiation of M. bovis from M. bovis BCG but not for differentiation among M. bovis strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of M. bovis between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of M. bovis genomic profiles. Our data showed the presence of a common M. bovis genotype in both cattle and wild boars, confirming the possible interspecies transmission of M. bovis.     

Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units

A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.     

Evolution and clonal traits of Mycobacterium tuberculosis complex in Guinea-Bissau

Two hundred twenty-nine consecutive isolates of Mycobacterium tuberculosis complex from patients with pulmonary tuberculosis in Guinea-Bissau, which is located in West Africa, were analyzed for clonal origin by biochemical typing and DNA fingerprinting. By using four biochemical tests (resistance to thiophene-2-carboxylic acid hydrazide, niacin production, nitrate reductase test, and pyrazinamidase test), the isolates could be assigned to five different biovars. The characteristics of four strains conformed fully with the biochemical criteria for M. bovis, while those of 85 isolates agreed with the biochemical criteria for M. tuberculosis. The remaining 140 isolates could be allocated into one of three biovars (biovars 2 to 4) representing a spectrum between the classical bovine (biovar 1) and human (biovar 5) tubercle bacilli. By using two genotyping methods, restriction fragment length polymorphism analysis with IS6110 (IS6110 RFLP analysis) and spoligotyping, the isolates could be separated into three groups (groups A to C) of the M. tuberculosis complex. Group A (n = 95), which contained the majority of classical human M. tuberculosis isolates, had large numbers of copies of IS6110 elements (mean number of copies, 9) and a distinctive spoligotyping pattern that lacked spacers 33 to 36. Isolates of the major group, group B (n = 119), had fewer IS6110 copies (mean copy number, 5) and a spoligotyping pattern that lacked spacers 7 to 9 and 39 and mainly comprised isolates of biovars 1 to 4. Group C isolates (n = 15) had one to three IS6110 copies, had a spoligotyping pattern that lacked spacers 29 to 34, and represented biovar 3 to 5 isolates. Four isolates whose biochemical characteristics conformed with those of M. bovis clustered with the group B isolates and had spoligotype patterns that differed from those previously reported for M. bovis, in that they possessed spacers 40 to 43. Interestingly, isolates of group B and, to a certain extent, also isolates of group C showed a high degree of variability in biochemical traits, despite genotypic identity in terms of IS6110 RFLP and spoligotype patterns. We hypothesize that isolates of groups B and C have their evolutionary origin in West Africa, while group A isolates are of European descent.     

Molecular epidemiological studies of Mycobacterium bovis infections in humans and animals in Sweden.

Forty-nine isolates of Mycobacterium bovis from humans and animals in Sweden were analyzed by restriction fragment length polymorphism (RFLP) patterns probed by the insertion element IS6110. Most isolates had patterns indicating the presence of only one or two genomic copies of the IS6110 insertion element. This simple type of pattern was found in all human isolates. In contrast, isolates from M. bovis infections in five herds of farmed deer in Sweden showed a specific RFLP pattern with seven bands, indicating seven copies of the IS6110 sequence. In 1958, Sweden was declared free from M. bovis in cattle. However, in 1987, M. bovis was reintroduced with imported farmed deer, and since 1991, 11 outbreaks in deer herds, but not in other livestock or wildlife, have been diagnosed. Continued RFLP studies of the new Swedish M. bovis isolates can reveal possible transmission of this deer strain to other animals or humans.     

Restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolated from countries in the western pacific region

A total of 422 Mycobacterium tuberculosis isolates from eight countries were subjected to IS6110 and IS1081 DNA fingerprinting by means of restriction fragment analysis to characterize M. tuberculosis strains from each country. Chinese, Mongolian, Hong Kong, Filipino, and Korean isolates had comparatively more copies of IS6110 (proportion with eight or more copies; 95% +/- 5%), while Thai, Malaysian, and Vietnamese isolates had fewer copies (proportion with eight or more copies, 60% +/- 4%). We found a number of novel IS1081 types in this study. One IS1081 type was present in 56% of Filipino isolates, had a specific 6.6-kb PvuII fragment in its IS6110 DNA fingerprint, and was termed the "Filipino family." The IS1081 types of Thai isolates had interposing characteristics between the characteristics of northeastern Asian and southeastern Asian IS1081 types. A 1.3-kb single-copy IS6110 fragment was found only in Vietnamese M. tuberculosis isolates. Although M. tuberculosis isolates from each country had comparatively similar characteristics depending on the classification factor, each country's isolates showed characteristic DNA fingerprints and differed slightly from the isolates from the other countries in either the mode number of IS6110 copies or the distribution of IS1081 types.     

Characterization of a trinucleotide repeat sequence (CGG)5 and potential use in restriction fragment length polymorphism typing of Mycobacterium tuberculosis

The genomes of 28 bacterial strains, including mycobacterial species Mycobacterium tuberculosis and Mycobacterium bovis, were analyzed for the presence of a special class of microsatellite, that of trinucleotide repeat sequences (TRS). Results of a search of all 10 possible TRS motifs (i.e., CCT, CGG, CTG, GAA, GAT, GTA, GTC, GTG, GTT, and TAT) with five or more repeating units showed that (CGG)(5) was highly represented within the genomic DNA of M. tuberculosis and M. bovis. Most of the (CGG)(5) repeats in the genome were within the open reading frames of two large gene families encoding PE_PGRS and PPE proteins that have the motifs Pro-Glu (PE) and Pro-Pro-Glu (PPE). (CGG)(5)-probed Southern hybridization showed that some mycobacterial species, such as Mycobacterium marinum, Mycobacterium kansasii, and Mycobacterium szulgai, possess many copies of (CGG)(5) in their genomes. Analysis of clinical isolates obtained from Tokyo and Warsaw with both IS6110 and (CGG)(5) probes showed that there is an association between the fingerprinting patterns and the geographic origin of the isolates and that (CGG)(5) fingerprinting patterns were relatively more stable than IS6110 patterns. The (CGG)(5) repeat is a unique sequence for some mycobacterial species, and (CGG)(5) fingerprinting can be used as an epidemiologic method for these species as well as IS6110 fingerprinting can. If these two fingerprinting methods are used together, the precise analysis of M. tuberculosis isolates will be accomplished. (CGG)(5)-based fingerprinting is particularly useful for M. tuberculosis isolates with few or no insertion elements and for the identification of other mycobacterial species when informative probes are lacking.     

Usefulness of Spoligotyping To Discriminate IS6110 Low-Copy-Number Mycobacterium tuberculosis Complex Strains Cultured in Denmark

Mycobacterium tuberculosis complex strains cultured in Denmark have been analyzed by IS6110 restriction fragment length polymorphism (RFLP) on a routine basis from 1992 and onwards. Due to the influx of immigrants with tuberculosis, the number of strains harboring only one to five copies of IS6110 has increased steadily. Since the discriminatory power of IS6110 fingerprinting for such strains is poor, we have performed additional genotyping of all low-copy-number strains by the recently described PCR-based method known as spoligotyping. A total of 311 clinical strains were typed: 14 Mycobacterium bovis BCG, 48 M. bovis, and 249 M. tuberculosis strains. Spoligotyping correctly differentiated M. bovis and M. bovis BCG from M. tuberculosis strains, but it did not differentiate M. bovis from M. bovis BCG. All M. bovis BCG strains exhibited identical spoligotype patterns. The discriminatory power of spoligotyping of low-copy-number M. tuberculosis strains was higher than that of IS6110 fingerprinting. Based on RFLP typing solely, 83% of the low-copy-number M. tuberculosis strains were found to form part of a cluster, and 75% were found to form a cluster on the basis of spoligotyping. When the two techniques were combined, the amount of clustering decreased to 55%. The combination of these two techniques might be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of the IS6110 element.     

Molecular epidemiology of Mycobacterium bovis in Texas and Mexico.

Seventy-nine Mycobacterium bovis isolates recovered from Mexican and Texas cattle were categorized into 16 and 25 distinct types on the basis of IS6110 and direct-repeat fingerprint patterns, respectively. By using a combination of both fingerprint patterns, 30 distinct restriction fragment length polymorphism types were defined. Fifty-eight of 79 isolates (73%) were distributed among nine clusters. Clustered isolates were identified within herds, as well as in geographically disperse herds in Texas and Mexico. This observation is consistent with active transmission within herds and among herds, presumably as a result of active or historical cattle movements. The majority of bovine isolates (64 of 79) exhibited a single copy of IS6110. Interestingly, in contrast to previous studies, a high percentage of bovine isolates (15 of 79) exhibited multiple IS6110 copies (two to five) distributed among 11 different restriction fragment length polymorphism types. It is speculated that transmission from noncattle sources may be responsible. Continued fingerprinting of isolates originating from nonbovine sources and herd surveys is expected to provide useful information regarding the epidemiology of tuberculosis in this region.     

Fast ligation-mediated PCR, a fast and reliable method for IS6110-based typing of Mycobacterium tuberculosis complex

IS6110 restriction fragment length polymorphism (RFLP) analysis is the most widely applied method for strain differentiation of Mycobacterium tuberculosis complex. We have previously described mixed-linker PCR, an IS6110-based PCR method that favorably compared with other typing methods for M. tuberculosis complex according to reproducibility and ability to differentiate between strains. Here we report the further development of this method, called fast ligation-mediated PCR (FLiP), which allows analysis of strains within one working day and starting from less than 1 ng of mycobacterial DNA or a crude cell lysate. Blinded analysis of a standard set of 131 M. tuberculosis complex and nontuberculous isolates showed the ability to differentiate 81 types among 90 M. tuberculosis complex isolates with 84 different IS6110 RFLP fingerprint patterns and detected 97% of the 31 duplicate samples. We suggest that FLiP can serve to rapidly detect chains of transmission prior to starting high-throughput analysis or standard IS6110 RFLP. It may as well serve as a secondary typing technique for other, non-IS6110-based methods.     

Stability of variable-number tandem repeats of mycobacterial interspersed repetitive units from 12 loci in serial isolates of Mycobacterium tuberculosis

Variable number tandem repeats (VNTRs) of elements named mycobacterial interspersed repetitive units (MIRUs) have previously been identified in 12 minisatellite loci of the Mycobacterium tuberculosis genome. These markers allow reliable high-throughput genotyping of M. tuberculosis and represent a portable approach to global molecular epidemiology of M. tuberculosis. To assess their temporal stability, we genotyped 123 serial isolates, separated by up to 6 years and belonging to a variety of distinct IS6110 restriction fragment length polymorphism (RFLP) families, from 56 patients who had positive sputum cultures. All 12 MIRU VNTR loci were completely identical within the groups of serial isolates in 55 out of 56 groups (98.2%), although 11 pairs of isolates from the same patients with conserved MIRU VNTRs displayed slightly different IS6110 RFLP profiles. In a single case, serial isolates with an unchanged IS6110 RFLP profile showed a change in 1 out of 12 MIRU VNTR loci. These results indicate that MIRU VNTRs are stable over time and therefore are suitable for reliable follow-up of patients chronically infected with tuberculosis over long periods. Moreover, they support MIRU VNTR genotyping as a powerful first-line method followed by subtyping by IS6110 RFLP to define ongoing transmission clusters.     

Study of Restriction Fragment Length Polymorphism Analysis and Spoligotyping for Epidemiological Investigation of Mycobacterium bovis Infection

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.     

Analysis of the regions responsible for IS6110 RFLP in a single Mycobacterium tuberculosis strain.

A high degree of IS6110 restriction fragment length polymorphism (RFLP) is observed amongst the different strains of the Mycobacterium tuberculosis complex. The sequences of the IS6110 flanking regions from a M. tuberculosis strain harbouring four IS6110 copies were determined. Duplication of 3-4 nucleotides was found at the extremities of the four IS6110 copies, suggesting that IS6110 RFLP is due to transposition of the IS element. One of the copies of IS6110 analysed in the study was shown to be located at the same site in the genome of M. tuberculosis as the single copy present in an M. bovis BCG strain.     

Characterization of IS6110 insertions in the dnaA–dnaN intergenic region of Mycobacterium tuberculosis clinical isolates

Mycobacterium tuberculosis isolates with identical IS 6110 restriction fragment length polymorphism (RFLP) patterns are considered to be clonally related. The presence of IS 6110 in the dnaA–dnaN intergenic region, one preferential locus for the integration of IS 6110 , was evaluated in 125 M. tuberculosis isolates. Five isolates had IS 6110 inserted in this region, and two consisted of a mix of isogenic strains that putatively have evolved during a single infection. Strains from the same isolate had identical spoligo and mycobacterial interspersed repetitive unit–variable-number tandem repeat profiles, but had slight variations in IS 6110 RFLP patterns, due to the presence of IS 6110 in the dnaA–dnaN intergenic region. Duplication of the dnaA–dnaN intergenic region was found in one isogenic strain.